Disruption of chromosome 11 in canine fibrosarcomas highlights an unusual variability of CDKN2B in dogs

BioMed CentralBMC Veterinary Research

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Open AcceResearch articleDisruption of chromosome 11 in canine fibrosarcomas highlights an unusual variability of CDKN2B in dogsJesús Aguirre-Hernández*, Bruce S Milne, Chris Queen, Patricia CM O'Brien, Tess Hoather, Sean Haugland, Malcolm A Ferguson-Smith, Jane M Dobson and David R Sargan
Address: Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, UK

Email: Jesús Aguirre-Hernández* - [email protected]; Bruce S Milne - [email protected]; Chris Queen - [email protected]; Patricia CM O'Brien - [email protected]; Tess Hoather - [email protected]; Sean Haugland - [email protected]; Malcolm A Ferguson-Smith - [email protected]; Jane M Dobson - [email protected]; David R Sargan - [email protected]

* Corresponding author

AbstractBackground: In dogs in the western world neoplasia constitutes the most frequently diagnosedcause of death. Although there appear to be similarities between canine and human cancers, ratherlittle is known about the cytogenetic and molecular alterations in canine tumours. Different dogbreeds are susceptible to different types of cancer, but the genetic basis of the great majority ofthese predispositions has yet to be discovered. In some retriever breeds there is a high incidenceof soft tissue sarcomas and we have previously reported alterations of chromosomes 11 and 30 intwo poorly differentiated fibrosarcomas. Here we extend our observations and present a casereport on detail rearrangements on chromosome 11 as well as genetic variations in a tumoursuppressor gene in normal dogs.

Results: BAC hybridisations on metaphases of two fibrosarcomas showed complexrearrangements on chromosome 11, and loss of parts of this chromosome. Microsatellite markerson a paired tumour and blood DNA pointed to loss of heterozygosity on chromosome 11 in theCDKN2B-CDKN2A tumour suppressor gene cluster region. PCR and sequencing revealed thehomozygous loss of coding sequences for these genes, except for exon 1β of CDKN2A, which codesfor the N-terminus of p14ARF. For CDKN2B exon 1, two alleles were observed in DNA from blood;one of them identical to the sequence in the dog reference genome and containing 4 copies of a 12bp repeat found only in the canine gene amongst all species so far sequenced; the other allele wasshorter due to a missing copy of the repeat. Sequencing of this exon in 141 dogs from 18 differentbreeds revealed a polymorphic region involving a GGC triplet repeat and a GGGGACGGCGGCrepeat. Seven alleles were recorded and sixteen of the eighteen breeds showed heterozygosity.

Conclusion: Complex chromosome rearrangements were observed on chromosome 11 in twoLabrador retriever fibrosarcomas. The chromosome alterations were reflected in the loss ofsequences corresponding to two tumour suppressor genes involved in cell-cycle progression.Sequencing of CDKN2B across many different breeds revealed a widespread polymorphism withinthe first exon of the gene, immediately before the ankyrin coding sequences.

Published: 31 July 2009

BMC Veterinary Research 2009, 5:27 doi:10.1186/1746-6148-5-27

Received: 1 May 2009Accepted: 31 July 2009

This article is available from: http://www.biomedcentral.com/1746-6148/5/27

© 2009 Aguirre-Hernández et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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BackgroundNeoplasia is profoundly important as a cause of morbid-ity and mortality in the domestic dog, Canis lupus famil-iaris. In addition to its importance as a working animaland a pet species, the dog is an important intermediatemodel species for human tumour biology. It has a rela-tively large body size, often displays responses to cytotoxicor other therapeutic agents comparable to humans, andhas a relatively high natural incidence of several cancerswith similar biology to human tumours. Dogs have beenused as models for cancer therapy, such as in osteosar-coma, in the giant dog breeds, oral melanoma and non-Hodgkins lymphoma, amongst others (reviewed in [1,2].In general, the same tumour types are recognised inhumans and dogs; for instance, the classification of softtissue sarcomas of dogs largely follows the human system[3]. Studies of cytogenetic and genetic changes in caninesoft tissue sarcomas may reveal more about their aetiol-ogy.

In a previous study, primary cell cultures were obtainedfrom two poorly differentiated fibrosarcomas [4]. Bothtumours (which were name-coded LE and ME) came fromadult female Labrador retrievers. Chromosome paintingrevealed they both had abnormalities in chromosomes 11(CFA11) and 30 (CFA30). One tumour (LE) containedfour translocation chromosomes involving CFA4, CFA11,CFA27 and CFA30 (tumour karyotype 2n = 78; t(4;11;30),t(11;27); t(27;11); t(30;4); der 11 (del 11q)); the secondone (ME) had a deletion of CFA11q and trisomy of CFA30(tumour karyotype 2n = 79; der11; +30). An attempt toisolate the translocation chromosomes by FluorescenceActivated Chromosome Sorting had not allowed com-plete purification of these chromosomes but PCR ofsequence tagged sites (STS), on sorted preparations, hadshown that sequences in the first 7 Mb of CFA 27 and thelast 20 Mb of CFA11 were present in the LE tumourgenome, but missing when t(27;11) was excluded. Rear-rangement in the Transforming Growth Factor BetaReceptor 1 gene (TGFBR1) was implied as only a portionof the exonic sequences could be obtained by PCR of DNAin this preparation. In addition to this altered copy ofTGFBR1, the tumour still had at least one normal codingsequence of the gene. This tumour sequence was identicalto that obtained from blood DNA of the same individual.At least one normal coding sequence of TGFBR1 was alsopresent in the tumour ME. Here we extend our search formolecular alterations of CFA11 that could be linked tocanine sarcomas and present a case report involving theloss of heterozygosity in a region harbouring two tumoursuppressor genes.

ResultsFurther characterisation of derivative chromosomes by BAC hybridisationTo obtain a more detailed description of rearrangementsin chromosome 11 in these fibrosarcomas, eleven canineBACs spaced at intervals along chromosome 11 were usedto probe tumour metaphases, in some cases in combina-tion with chromosome paints identifying translocationpartners (Table 1). For LE, the distribution of BAC hybrid-isation suggests that the der11 and t(4;11;30) chromo-somes originate from one chromosome 11 whilstt(11;27) and t(27;11) originate from the other (Figure 1).However, the pattern of rearrangements in derivativechromosomes is complex. The der11 chromosome con-tains both centromeric and telomeric sequences in aninverted arrangement, whilst central parts of this chromo-some 11 are found in t(4;11;30) (BACs between 375-F21and 381-N7 locate here). t(11;27) contains the centro-mere proximal region of CFA11. Although no BACs moretelomeric than 375-F21 hybridised to this chromosome,chromosome painting suggests that more than half of thenormal CFA11 is present, so there may be some furtherinterstitial insertion of sequences not represented by theBACs used here. More telomeric BACs hybridised tot(27;11) but, again, these showed rearrangement suchthat 381-H22 has a sub-telomeric position whilst BACswhich lie distal to this in the normal chromosome arenow placed near the centre of the derivative. The translo-cation break/fusion point is close to RP81_381-N7 withBACs derived from centromere proximal sequence founddistal to this position in the fusion chromosome.

For ME, BACs telomeric of RP81_381-H22 did not hybrid-ise to the derivative chromosome, but RP81_381-H22itself did (Figure 2). There was also lack of signal fromRP81_376-M15 on the derivative, suggesting interstitialdeletion of the derivative chromosome in the area of thisBAC. In independent RH mapping studies the BAC 381-F14 is placed on chromosome 11 at position 8810, telom-eric to 373M14 [5].

Loss of heterozygosityFor LE, genomic DNA from blood and tumour was avail-able, while only tumour DNA could be obtained for sam-ple ME. Loss of heterozygosity on CFA11 was analysed forthe LE fibrosarcoma. Forty-five microsatellites distributedacross the chromosome were studied (Table 2). Completeloss of heterozygosity (loss of the 262 bp allele) wasobserved for marker CAMC11.029, and a substantial loss(of the 224 bp allele) for marker CAMC11.026 (22%remaining signal) (see Figure 3). The region between thesetwo markers contains sequences orthologous to thehuman genes coding for the proteins p15INK4B, p16INK4A

and p14ARF. The first one is coded by the cyclin-dependentkinase inhibitor 2B gene (CDKN2B), while the other two



are coded by cyclin-dependent kinase inhibitor 2A(CDKN2A) [reviewed in [6]]. A marker (CAMC11.027)within the canine CDKN2B-CDKN2A region was unin-formative (homozygous in blood and tumour DNA forsample LE). Most other markers showed no changebetween tumour and blood, but one further marker,(FH4031, 29.9 Mb proximal from CAMC11.026), showedpartial loss of one allele (37% remaining signal). Formarker CAMC11.004 (10 Mb from CAMC11.026) a dis-crepancy was observed in the results corresponding to theblood and the tumour; the former presented a hetero-zygous genotype, while the tumour had a single allele thatwas different to either of those observed in the blood.

CDKN2B and CDKN2A sequencing from tumour DNACDKN2A and CDKN2B have not been fully described inthe dog. No mRNAs or ESTs that completely define thesegenes are available, and gene prediction programmes donot fully agree with each other or with those EST that areavailable. In addition, the area is extremely GC rich andhas proved difficult to clone or to sequence, containing agap in the CamFam 2.0 genome assembly [7,8]. Align-ments with human and other CDKN2A/B RNAs appear tobe the best guide to gene structure. We used these todesign PCR primers. To determine whether the expectedremaining copy of these genes had any mutations intumour LE, the predicted exonic sequences, and theirflanking intronic sequences, were investigated. Since the

gap in the dog reference sequence is where CDKN2A exon1α is expected to be present (Table 2 and Figure 3), it wasnot studied. However, a PCR product could be obtainedfor the predicted exon 1β; this sequence was identical tothat observed in blood DNA from the same individual,and to the sequence in the reference genome. In contrastto this, for CDKN2A no PCR products of exon 2 could beobtained from tumour LE's DNA in spite of repeatedattempts, but the sequence was obtained from blood DNAand it contained no difference when compared to the ref-erence genome.

When using DNA from tumour as template, no PCR prod-ucts could be obtained for CDKN2B exons 1 and 2 andtheir flanking intronic sequences. In LE's DNA fromblood, the sequence of CDKN2B exon 2 was the same asin the reference genome. Exon 1 of this same gene couldnot be PCR-amplified from tumour LE but it was ampli-fied using DNA from blood, and then cloned andsequenced. The results showed that this individual washeterozygous, having one allele with the same sequence asthe reference genome (g. [109GGC[5];124GGGGACGCCGCC[4]]) as well as a shorter allele (g.[109GGC[5]; 124GGGGACGCCGCC[3]]). At the aminoacid level the first allele would have five Gly residues fol-lowed by four tandem copies of the sequence GlyAspGly-Gly (p. [Gly10[5]; Gly15_Gly18[4]]), while the secondallele would consist of five Gly followed by only 3 copies

Table 1: BACs used for in situ hybridisation.

BAC name CFA Genomic position(bp)

Signal on LEder11

Signal on LEt(4;11;30)

Signal on LEt(11;27)

Signal on LEt(27;11)

Signal on MEder11

386-H9 11 6153594 + + +

385-C1 11 22151575 NA* NA NA +

375-F21 11 24354423 + + +

372-O3 11 32432415 + + +

372-K9 11 33384887 NA NA NA +

381-N7 11 41509254 + + +

376-M15 11 46649071 NA NA NA -

381-H22 11 49355967 + + +

373-C5 11 59457191 + + -

373-M14 11 65702272 NA NA NA -

381-F14 27 38704742 + + -

*NA = Data not available.



of the tetrapeptide (p. [Gly10[5]; Gly15_Gly18[3]]) (Fig-ure 4).

CDKN2B exon 1 polymorphism in the dogTo investigate whether the shorter allele could be relatedto the development of the neoplasia, the exon was wasamplified using DNA from blood of 141 dogs of 18 differ-ent breeds. The size of the PCR products was determinedby capillary electrophoresis and products correspondingto the different sizes were sequenced directly (Figure 5).Seven different alleles were found (see Figure 6, and Addi-tional file 1: Distribution of CDKN2B exon 1 alleles invarious dog breeds). The most frequent allele was p.[Gly10[5]; Gly15_Gly18[4]], which corresponds to thereference sequence and was found in all breeds studied.Alleles p. [Gly10[5]; Gly15_Gly18[3]] and p. [Gly10[4];Gly15_Gly18[4]] were also observed. Additionally, 4 alle-les were rarer in this sample set; the shortest being p.[Gly10[4]; Gly15_Gly18[2]] in Cardigan Welsh corgis,and the longest p. [Gly10[5]; Gly15_Gly18[5]] in a Rott-weiler. Among these blood samples, 24 corresponded toflat-coated retrievers; of these 11 had histiocytic sarcoma

and 13 were non-affected dogs aged 10 years or more (SeeAdditional file 1: Distribution of CDKN2B exon 1 allelesin various dog breeds). The distribution of the alleles wasnot significantly different between these two sets (chi-square test p = 0.55). All breeds except boxer (15 individ-uals examined) and Shih Tzu (two individuals only) werepolymorphic at this position

Other CDKN2B and CDKN2A polymorphisms in the dogTo search for additional polymorphisms in these twogenes, exon 2 of CDKN2B was studied using DNA fromblood samples from a Bernese mountain dog, four boxers,nine flat-coated retrievers (four with histiocytic sarcomaand five free from neoplasia), two golden retrievers and aLabrador retriever. No polymorphisms were seen in thecoding region or the flanking intronic bases.

Exon 2 of CDKN2A was also sequenced from DNAobtained from blood samples from three Bernese moun-tain dogs, four flat-coated retrievers with histiocytic sar-coma, and five flat-coated retrievers free from tumours,two golden retrievers and four Labrador retrievers. A cod-

Chromosome painting of metaphases spreads from canine fibrosarcoma LEFigure 1Chromosome painting of metaphases spreads from canine fibrosarcoma LE. Chromosome painting of tumour LE (6 colour paints, colours as shown) shows 4 derivative chromosomes containing CFA11 DNA. To look at the distribution of CFA11 BACs, metaphases were hybridised with a mixed paint of CFA 4, 27 and 30 (green) and a single BAC paint as indicated in examples shown, (red). For N7, where the t(27;11) is difficult to identify because of a high green background, additional hybridising chromosomes from other metaphases are shown; inset at twice the scale. Diagrams summarise hybridisation of all BACs with each derivative containing chromosome 11. The red arrowheads denote BACs consistently binding to the chromo-somes shown. Chromosomes in the diagram are CFA11 in blue (mid blue -centromeric; light blue -telomeric); CFA27 green; CFA4 yellow; CFA30 purple.



ing SNP was identified that led to a non-synonymouschange in the predicted codon 19 of exon 2 that is sharedby both p16INK4A and p14ARF. The polymorphism was ag.1206A>G transversion that corresponds to a p.Gln79Argchange in the predicted amino acid sequence of p14ARF

(Figure 7) and an Asn to Asp change in the p16INK4A read-ing frame (Figure 8). Both alleles were found in all breedsstudied, except in the boxer where all four individualswere homozygous for the reference sequence. In the flat-coated retrievers half of the histiocytic sarcoma cases wereGG homozygous and the rest AG heterozygous, while twocontrols were homozygous GG and three were hetero-zygous.

DiscussionA previous analysis of two canine fibrosarcomas with rear-rangement of chromosome 11 was followed up by BACmapping. In both tumours this revealed that rearrange-ments were more complex than had been seen using chro-mosome painting. In LE all chromosomes containing

fragments of chromosome 11, except t(11;27), showedinterstitial deletions and or rearrangements. In ME a dele-tion around BAC M15 was observed. Loss of heterozygos-ity (LOH) on chromosome 11 was studied in LE's tumour.Marker CAMC011.004 (34.56 Mb) presented an allele inthe tumour which was different from any of those in theblood. This result was confirmed in two independentexperiments. This marker is within a gene desert, with noproven transcripts encoded or genes modelled withinabout a megabase. Our data does not show whether thismarker is close to a rearrangement. FH4031 (14.24 Mb),which shows LOH, is also some way from the nearestknown gene. In this case more proximal markers are notinformative. It seems likely that, in the tumour, the der11and t(4;11;30) chromosomes have lost some sequences inthis region, which is near the proximal chromosomebreakpoint in both.

Loss of heterozygosity was also observed in two markerswhich bracket a region encompassing the CDKN2B-

Chromosome painting of metaphases spreads derived from fibrosarcoma MEFigure 2Chromosome painting of metaphases spreads derived from fibrosarcoma ME. Chromosome painting of tumour ME (6 colour paints, colours as shown) shows a der11 with ± 40% of the genomic material of the normal chromosome. An exam-ple BAC hybridisation is shown, using RP81_381-N7 (red). Diagrams of the normal and derivative chromosome show hybridi-sation positions of BACs: red arrowheads – BACs consistently binding to both chromosomes; green arrowheads -those not hybridising to the derivative; blue arrowhead -consistent hybridisation to the normal but inconsistent to the der 11 chromo-some.



Table 2: CFA11 loss of heterozygosity.

Feature* Sequence type Mb Distance From Top LE blood genotype§ LE tumour genotype Observations

CAMC11.009 Microsatellite 4.985028 387, 387 387, 387

AHT137 Microsatellite 5.829562 148, 148 148, 148

REN161P13 Microsatellite 7.106779 189, 189 189, 189


FH3203 Microsatellite 8.623136 89, 89 89, 89


FH4031 Microsatellite 14.237434 312, 331 312, 331 LOH

REN286P10 Microsatellite 17.844923 182, 185 182, 185

REN242K04 Microsatellite 19.23201 328, 328 328, 328



REN142O09 Microsatellite 26.338024 256, 261 256, 261














CAMC11.026 Microsatellite 44.172674 224, 226 224, 226 LOH

CDKN2A Gene, exon 2 44.255474 Present Missing

Contig_16328 contig end 44.258553



Gap in dog sequence 44.258554

CDKN2A Gene, exon 1α Undetermined

Gap in dog sequence 44.260435

Contig_16329 contig start 44.260436

CDKN2A Gene, exon 1β 44.280332 Present Present


CDKN2B Gene, exon 2 44.291018 Present Missing

CDKN2B Gene, exon 1 44.293951 Heterozygous Missing

CAMC11.029 Microsatellite 44.304276 256, 262 256, 256 LOH


C11.868 Microsatellite 48.547399 216, 216 216, 216






REN249L05 Microsatellite 58.631801 175, 175 175, 175

TGFBR1 Gene 59.220624

CAMC11.024 Microsatellite in TGFRB1 intron 7 59.239311 310, 310 310, 310



REN147O02 Microsatellite 61.367981 243, 243 243, 243



C11.873 Microsatellite 67.767854 136, 144 136, 144


DGN13 Microsatellite 72.807881 303, 326 303, 326

*Microsatellites are listed according to their position in Ensembl's CanFam 2.0 (July 2008). The position of exons within the CDKN2B-CDKN2A gene cluster is provided, along with the position of the gap between contigs 16328 and 16329, and the location of TGFBR1. §The numbers in the genotype columns correspond to the PCR product sizes (in base pairs) of the alleles, as determined by the sequencing instrument.

Table 2: CFA11 loss of heterozygosity. (Continued)



CDKN2A gene cluster. Even though the microsatellitestudy pointed to loss of heterozygosity rather than com-plete loss of the region containing CDKN2A andCDKN2B, no PCR products could be obtained from thetumour for the genes themselves, except for CDKN2Aexon 1β. The sequence of this exon showed no differenceswith the reference one. A microsatellite within the genecluster could be amplified but it was homozygous inblood and thus non-informative regarding loss of hetero-zygosity. This microsatellite is 8 kb away from CDKN2Aexon 1β and 3 kb from CDKN2B exon 2, which could notbe detected in the tumour DNA. These results suggest thattwo copies of parts of chromosome 11 are present in thetumour, while other regions are present only once, andstill others, close to the latter, are completely missing. Thiscomplex pattern is reminiscent of the alterations observedfor t(27;11), where TFGBR1 exons 3 to 6 and 9 wereobserved, but not the rest of the exons [4]. This agrees alsowith the recent observation on the complexity of chromo-some rearrangements in human tumours [9].

Both approaches, BAC mapping and loss of heterozygos-ity, point to alterations on canine chromosome 11, in theregion equivalent to human chromosome 9p21 which iscommonly deleted in human tumours [10] and containsthe genes CDKN2B and CDKN2A. The first codes for theprotein p15INK4B, while the second codes for p16INK4A andp14ARF which are proteins derived from alternate exon 1sequences and use different reading frames for the com-mon exon 2 [11,12]. p16INK4A and p14ARF regulate theretinoblastoma protein 1 (pp105RB1) and the tumour pro-tein 53 (TP53), respectively. These form part of the twopathways most commonly disrupted in human tumours.

Recently a new protein, smARF, also coded by CDKN2A,was identified in mouse and human and localised in themitochondria [13]. It has been shown to inhibit cellgrowth and proliferation, and to induce apoptosis in aTP53-independent manner [14,15]. CDKN2A also codesfor the proteins p12 and p16γ which are less well under-stood [16,17]. In mice, disruption of either p19Arf (theorthologue of human p14ARF) or p16Ink4b, or both, resultsin increased predisposition to tumour development [18-21]. In humans, homozygous deletions are the most fre-quent type of mutation involving these genes [22], asopposed to the combination of mono-allelic deletion fol-lowed by the mutation of the remaining copy of the gene,which is the common pattern in other tumour suppressorgenes [23]. However, alterations affecting only one ofp15INK4B, p16INK4A or p14ARF have been reported [11].Apart from homozygous deletions, 5'CpG methylationhas also been observed in some tumours [12,24-26]. Inhuman patients with soft tissue sarcomas, loss ofCDKN2B and CDKN2A is associated with reduced sur-vival [27], while in dogs, absence or reduced levels ofp16INK4A have been reported in melanoma tumours andcell lines [28], as well as in osteosarcoma cell lines [29]. Incanine non-Hodgkin's lymphoma (NHL), deletion ofp16INK4A or loss of CFA11 have been observed in high-grade T-cell NHL, without comparable alterations seen inhigh-grade B-cell NHL or in low grade tumours [30]. Inthis same paper, p16INK4A methylation was observed in asingle low-grade T-cell lymphoma. In addition to this, intwo canine mammary tumour cell lines, CMT12 andCMT27, no expression of p16INK4A was observed, while ina third cell line (CMT28) increased expression wasdetected [31]. Upon transfection of a complete human

Map of the canine region containing the CDKN2B-CDKN2A locusFigure 3Map of the canine region containing the CDKN2B-CDKN2A locus. Top, position of CAMC11.026, CAMC11.029, and of the gap in the genomic sequence assembly. Middle, microsatellites presenting loss of heterozygosity; also, position of pre-dicted CDKN2 related exons (darker grey), other unrelated exons (light grey), and canine ESTs in the database (black). Bottom, exon designations used here.



p16INK4A cDNA, these cell lines lost most of the character-istics of the transformed phenotype [31]. The sequences ofCMT28 and of canine fibroblast cultured cells werereported to differ from the reference sequence, althoughthe nucleotide and the aminoacid sequences are identicalto the canine reference sequences used here for the secondexon of p16INK4A and p14ARF (Figure 7 and Figure 8).

CDKN2A exon 1β codes for the N-terminus portion ofp14ARF. The first amino-acid residues of this protein arerelatively well conserved and have an important role inbinding MDM2 while the rest of the protein, containing anucleolar localization signal, is poorly conserved and maybe dispensable [11,32,33]. In chicken, exon 1α of

CDKN2A has been lost and no p16INK4A protein is pro-duced [34]. Exon 1β is spliced to the still existent exon 2but the predicted protein terminates at the end of exon 1β,with no residues from exon 2. In spite of this, the proteinis able to bind MDM2 thus preventing the induction ofTP53 degradation and the inhibition of TP53 expression[34]. It is therefore likely that exon 1β codes for the aminoacid residues required for p14ARF to perform its normalrole. Moreover, aberrant p16INK4A transcripts have beenobserved in tumours [35] and some of them are translatedand are able to function in cell-cycle control [36]. In thepresent study only exon 1β of CDKN2A was observed inLE's fibrosarcoma. It is possible that an abnormal versionof p14ARF could have been produced; it is unknown

Predicted canine CDKN2B exon 1 amino acid sequencesFigure 4Predicted canine CDKN2B exon 1 amino acid sequences. Alternate Gly residues in the polymorphic region have a grey background, while the GlyAspGlyGly repeats are enclosed in a frame and alternate repeats also have a grey background. In LE's tumour DNA the exon was missing, but not in peripheral blood where it was heterozygous. Alleles are identified in a simplified way (e.g., [2][3]] corresponds to p. [Gly10[2];Gly15_Gly18[3]]).

01.Xenopus_tropicalis -------MAFNANT-----------------------LCSACARGDVDLA 20 02.Rattus_norvegicus -------ML-GGGS--------------------DAGLATAAARGQVETV 22 03.Mus_musculus -------ML-GGSS--------------------DAGLATAAARGQVETV 22 04.Bos_taurus -------MLSGGGG--------------------DADLANAAARGQVEAV 23 05.Macaca_mulatta MREENKGMPSGGGS--------------------DEGLTSAAARGLVEKV 30 06.Pan_troglodytes MREENKGMPSGGGS--------------------DEGLASAAARGLVEKV 30 07.Homo_sapiens MREENKGMPSGGGS--------------------DEGLASAAARGLVEKV 30 08.[[4];[2]] MREEATGLLGGGG-------------GDGGGDGGDAGLASASAQGQAETV 37 09.[[5];[2]] MREEATGLLGGGGG------------GDGGGDGGDAGLASASAQGQAETV 38 10.[[2];[3]] MREEATGLLGG-----------GDGGGDGGGDGGDAGLASASAQGQAETV 39 11.[[5];[3]] MREEATGLLGGGGG--------GDGGGDGGGDGGDAGLASASAQGQAETV 42 12.[[4];[4]] MREEATGLLGGGG-----GDGGGDGGGDGGGDGGDAGLASASAQGQAETV 45 13.[[5];[4]] CanFam2.0 MREEATGLLGGGGG----GDGGGDGGGDGGGDGGDAGLASASAQGQAETV 46 14.[[5];[5]] MREEATGLLGGGGGGDGGGDGGGDGGGDGGGDGGDAGLASASAQGQAETV 50 15.LE_blood01 MREEATGLLGGGGG----GDGGGDGGGDGGGDGGDAGLASASAQGQAETV 46 16.LE_blood01 MREEATGLLGGGGG--------GDGGGDGGGDGGDAGLASASAQGQAETV 42 01.Xenopus_tropicalis RQMLQSGIPVNATNSHGRTPIQ 42 02.Rattus_norvegicus RQLLEAGADPNAVNRFGRRPIQ 44 03.Mus_musculus RQLLEAGADPNALNRFGRRPIQ 44 04.Bos_taurus RQLLEAGVDPNRLNRFGRRPIQ 45 05.Macaca_mulatta RQLLEAGADPNGVNRFGRRAIQ 52 06.Pan_troglodytes RQLLEAGADPNGVNRFGRRAIQ 52 07.Homo_sapiens RQLLEAGADPNGVNRFGRRAIQ 52 08.[[4];[2]] RQLLEAGADPNGVNSFGRRPIQ 59 09.[[5];[2]] RQLLEAGADPNGVNSFGRRPIQ 60 10.[[2];[3]] RQLLEAGADPNGVNSFGRRPIQ 61 11.[[5];[3]] RQLLEAGADPNGVNSFGRRPIQ 64 12.[[4];[4]] RQLLEAGADPNGVNSFGRRPIQ 67 13.[[5];[4]] CanFam2.0 RQLLEAGADPNGVNSFGRRPIQ 68 14.[[5];[5]] RQLLEAGADPNGVNSFGRRPIQ 72 15.LE_blood01 RQLLEAGADPNGVNSFGRRPIQ 68 16.LE_blood01 RQLLEAGADPNGVNSFGRRPIQ 64




CDKN2B exon 1 genotype determination in dogs of different breedsFigure 5CDKN2B exon 1 genotype determination in dogs of different breeds. Dye-labelled PCR products were separated by capillary electrophoresis. Fragments representing all different sizes were sequenced to confirm the identity of the alleles.

Great dane [[5];[3]] Bassett hound [[2];[3]] [[5];[3]]

350 375 400Size (nt)

350 375 400Size (nt)

German shepherd dog [[5];[3]] Cairn terrier [[5];[2]] [[5];[4]]

350 375 400Size (nt)

350 375 400Size (nt)

Flatcoated retriever [[4];[4]] Labrador retriever [[5];[3]] [[5];[4]]

350 375 400Size (nt)

350 375 400Size (nt)

Boxer [[5];[4]] Newfoundland [[5];[3]] [[4];[4]]

350 375 400Size (nt)

350 375 400Size (nt)

Cardigan Welsh corgi [[4];[2]] [[5];[3]] Rottweiler [[5];[3]] [[5];[5]]

350 375 400Size (nt)

350 375 400Size (nt)

Flatcoated retriever [[4];[4]] [[5];[4]]

350 375 400Size (nt)



CDKN2B exon 1 alleles observed in canine blood and tumour samplesFigure 6CDKN2B exon 1 alleles observed in canine blood and tumour samples. Alternate GGC triplets coding for Gly have a grey background. The dodecameric nucleotide sequence (GGGGACGGCGGC) coding for the GlyAspGlyGly repeats are within a box, with alternate repeats having a grey background. Intronic sequences are in lowercase. Alleles are identified with a short version of their description; e.g., [4][2]] stands for g. [109GGC[4];124GGGGACGGCGGC[2]]. The [5][4]] allele corre-sponds to the reference genome (CanFam2.0).

[[4];[2]] ------------------------------------------------cctcccggcccg 12 [[5];[2]] ---------------------------------------------------cccggcccg 9 [[2];[3]] ------------------------------------------------------------ [[5];[3]] ---------------------------------------------------cccggcccg 9 [[4];[4]] ------------------------------------------------------------ [[5];[4]] gaaacagggaggcgagtgacgaagctccgctcacggcccgcggtggatcctcccggcccg 60 [[5];[5]] ------------------------------------------------------------ [[4];[2]] tccgcgtctgcgggctgcgagATGCGCGAGGAGGCCACGGGCTTGCTCGGCGGCGGCGGC 72 [[5];[2]] tccgcgtctgcgggctgcgagATGCGCGAGGAGGCCACGGGCTTGCTCGGCGGCGGCGGC 69 [[2];[3]] ------------ggctgcgagATGCGCGAGGAGGCCACGGGCTTGCTCGGCGGC------ 42 [[5];[3]] tccgcgtctgcgggctgcgagATGCGCGAGGAGGCCACGGGCTTGCTCGGCGGCGGCGGC 69 [[4];[4]] ------tctgcgggctgcgagATGCGCGAGGAGGCCACGGGCTTGCTCGGCGGCGGCGGC 54 [[5];[4]] tccgcgtctgcgggctgcgagATGCGCGAGGAGGCCACGGGCTTGCTCGGCGGCGGCGGC 120 [[5];[5]] ---------------tgcgagATGCGCGAGGAGGCCACGGGCTTGCTCGGCGGCGGCGGC 45 *************************************** [[4];[2]] ---------------------------------------GGGGACGGCGGCGGGGACGGC 93 [[5];[2]] GGC------------------------------------GGGGACGGCGGCGGGGACGGC 93 [[2];[3]] ---------------------------GGGGACGGCGGCGGGGACGGCGGCGGGGACGGC 75 [[5];[3]] GGC------------------------GGGGACGGCGGCGGGGACGGCGGCGGGGACGGC 105 [[4];[4]] ---------------GGGGACGGCGGCGGGGACGGCGGCGGGGACGGCGGCGGGGACGGC 99 [[5];[4]] GGC------------GGGGACGGCGGCGGGGACGGCGGCGGGGACGGCGGCGGGGACGGC 168 [[5];[5]] GGCGGGGACGGCGGCGGGGACGGCGGCGGGGACGGCGGCGGGGACGGCGGCGGGGACGGC 105 ********************* [[4];[2]] GGCGACGCGGGCCTGGCCAGCGCCTCGGCGCAGGGGCAGGCGGAGACCGTGCGGCAGCTC 153 [[5];[2]] GGCGACGCGGGCCTGGCCAGCGCCTCGGCGCAGGGGCAGGCGGAGACCGTGCGGCAGCTC 153 [[2];[3]] GGCGACGCGGGCCTGGCCAGCGCCTCGGCGCAGGGGCAGGCGGAGACCGTGCGGCAGCTC 135 [[5];[3]] GGCGACGCGGGCCTGGCCAGCGCCTCGGCGCAGGGGCAGGCGGAGACCGTGCGGCAGCTC 165 [[4];[4]] GGCGACGCGGGCCTGGCCAGCGCCTCGGCGCAGGGGCAGGCGGAGACCGTGCGGCAGCTC 159 [[5];[4]] GGCGACGCGGGCCTGGCCAGCGCCTCGGCGCAGGGGCAGGCGGAGACCGTGCGGCAGCTC 228 [[5];[5]] GGCGACGCGGGCCTGGCCAGCGCCTCGGCGCAGGGGCAGGCGGAGACCGTGCGGCAGCTC 165 ************************************************************ [[4];[2]] CTGGAAGCCGGCGCGGATCCCAACGGAGTCAACAGCTTCGGCAGGCGCCCGATCCAGgta 213 [[5];[2]] CTGGAAGCCGGCGCGGATCCCAACGGAGTCAACAGCTTCGGCAGGCGCCCGATCCAGgta 213 [[2];[3]] CTGGAAGCCGGCGCGGATCCCAACGGAGTCAACAGCTTCGGCAGGCGCCCGATCCAGgta 195 [[5];[3]] CTGGAAGCCGGCGCGGATCCCAACGGAGTCAACAGCTTCGGCAGGCGCCCGATCCAGgta 225 [[4];[4]] CTGGAAGCCGGCGCGGATCCCAACGGAGTCAACAGCTTCGGCAGGCGCCCGATCCAGgta 219 [[5];[4]] CTGGAAGCCGGCGCGGATCCCAACGGAGTCAACAGCTTCGGCAGGCGCCCGATCCAGgta 288 [[5];[5]] CTGGAAGCCGGCGCGGATCCCAACGGAGTCAACAGCTTCGGCAGGCGCCCGATCCAGgta 225 ************************************************************ [[4];[2]] gctggggccccggggcctcgccggcagggggcgcaggaacgcaggggagcggcctccgtg 273 [[5];[2]] gctggggccccggggcctcgccggcagggggcgcaggaacgcaggggagcggcctccgtg 273 [[2];[3]] gctggggccccggggcctcgccggcagggggcgcaggaacgcaggggagcggcctccgtg 255 [[5];[3]] gctggggccccggggcctcgccggcagggggcgcaggaacgcaggggagcggcctccgtg 285 [[4];[4]] gctggggccccggggcctcgccggcagggggcgcaggaacgcaggggagcggcctcc--- 276 [[5];[4]] gctggggccccggggcctcgccggcagggggcgcaggaacgcaggggagcggcctccgtg 348 [[5];[5]] gctggggccccggggcctcgccggcagggggcgcaggaacgcaggggagcggcctcc--- 282 ********************************************************* [[4];[2]] ---------------------------------------- [[5];[2]] ggacgcgacggg---------------------------- 285 [[2];[3]] ggacgc---------------------------------- 261 [[5];[3]] ggacgcgacggg---------------------------- 297 [[4];[4]] ---------------------------------------- [[5];[4]] ggacgcgacggggaccgagagcttcgccgacgtatctgcg 388 [[5];[5]] ----------------------------------------


whether the remaining exon 1β would have been splicedto another sequence, generating an abnormal transcriptand, possibly, a shorter version of p14ARF, as is in thechicken. In the current study, a polymorphism wasobserved in exon 2 causing missense mutations in bothp16INK4A and p14ARF. The polymorphic residue is in theloop joining the second and thitd ankyrin repeats ofp16INK4A. This residue is on the surface of the protein [37].Although it does not interact with CDK6, it does seem tohave a role in stabilising the structure of p16INK4A [38,39].In other species this position is occupied by the acidic res-idues Asp or Glu, so the polar neutral Asn residue seemsto be unique to dogs. However, other structurally relatedproteins with ankyrin repeats have Asn in the equivalentposition (e.g., p18) [37].

Polymorphisms in short repeat sequences within codingexons have been observed before [40,41], whilst somaticrepeat instabilities are well documented in other species

both in tumours and in some degenerative diseases. Theputative first exon of CDKN2B in dogs has a predictedGlyAspGlyGly repeat that is not found in other species.This exon is represented in the database by two ESTs [Gen-Bank: CX985268 and GenBank: DN749168]. The multi-plicity of alleles in this gene may have resulted fromoccasional misalignments during crossover eventsbetween homologous chromosomes, leading to theexpansion and contraction in the number of GGC tripletand GGGGACGGCGGC dodecamer repeats. No instanceswere observed of nonsense mutations in this region, norwere indels found that would produce shifts in the read-ing frame. In the case of flat-coated retrievers, which showa high predisposition to developing histiocytic sarcomas,cases and controls were found to have a similar distribu-tion of exon 1 alleles. Moreover, the sequences obtainedfrom genetic material from blood samples of variousbreeds suggest that the variation in the number of Gly res-

Polymorphism in exon 2 of canine CDKN2A in the p14ARF reading frame and alignment to orthologous sequencesFigure 7Polymorphism in exon 2 of canine CDKN2A in the p14ARF reading frame and alignment to orthologous sequences. The dog polymorphic residue is highlighted in yellow.

Canine p14ARF and orthologous sequences Rattus_norvegicus GDDDGQRQSGSSPALLWCRFELRGPHHPLPTGARRSAGGLPRHSGSTAPG 50 Mus_sp. GDDDGQRSRSSSSAQLRCRFELRGPHYLLPPGARRSAGRLPGHAGGAARV 50 Mesocricetus_auratus GDDDGQHPSSQTAAALRCGTELRGPSHPLPTRARCSTGGLLGNSGDTTPG 50 BMD GHDDGQHPRGPAAAAPRRRPQLCRPRHPHPPCARRGPGGLPGHAGGAAPS 50 CanFam2.0 GHDDGQHPRGPAAAAPRRQPQLCRPRHPHPPCARRGPGGLPGHAGGAAPS 50 CMT28_FJ542309 GHDDGQHPRGPAAAAPRRQPQLCRPRHPHPPCARRGPGGLPGHAGGAA-- 48 NCF_FJ542308 GHDDGQHPRGPAAAAPRRQPQLCRPRHPHPPCARRGPGGLPGHAGGAA-- 48 Homo_sapiens GHDDGQRPSGGAAAAPRRGAQLRRPRHSHPTRARRCPGGLPGHAGGAAPG 50 *.****: . :.* :* * : *. ** .* * ::*.:: Rattus_norvegicus RGAAGCARCLGSPAARPGPRAGTSRRRAVFA----------VSTLLRWER 90 Mus_sp./ RGSAGCARCLGSPAARLGPRAGTSRHRAIFAFRWVLFVFRWVVFVYRWER 100 Mesocricetus_auratus GGAAGCA------------------------------------------- 57 BMD RGAAGRARCLGPPARGPG-------------------------------- 68 CanFam2.0 RGAAGRARCLGPPARGPG-------------------------------- 68 CMT28_ FJ542309 -------------------------------------------------- NCF_ FJ542308 -------------------------------------------------- Homo_sapiens RGAAGRARCLGPSARGPG-------------------------------- 68 *:** * Rattus_norvegicus FPGHRQA 97 Mus_sp. RP-DRRA 106 Mesocricetus_auratus ------- BMD ------- CanFam2.0 ------- CMT28_ FJ542309 ------- NCF_ FJ542308 ------- Homo_sapiens -------


http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=CX985268

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=DN749168



Polymorphism in exon 2 of canine CDKN2A in the p16INK4A reading frame and alignment to orthologous sequencesFigure 8Polymorphism in exon 2 of canine CDKN2A in the p16INK4A reading frame and alignment to orthologous sequences. The canine polymorphic position is highlighted in yellow in the loop joining the second and third ankyrin repeats. The α helix residues of the ankyrin repeats have a grey background. CanFam2.0 -dog reference genome; BMD -Bernese Moun-tain dog with the alternative Asp residue; CMT28_FJ542308 -canine mammary tumour cell line; NCF_FJ542309 -cultured canine fibroblasts.

Canine p16INK4A and orthologous sequences Helices 3 helix 4 helix 5 helix 6 Mus_spretus VMMMGNVHVAALLLNYGADSNCEDPTTFSRPVHDAAREGFLDTLVVLHGS 50 Mus_musculus VMMMGNVHVAALLLNYGADSNCEDPTTFSRPVHDAAREGFLDTLVVLHGS 50 Rattus_norvegicus VMMMGNVKVAALLLSYGADSNCEDPTTLSRPVHDAAREGFLDTLVVLHQA 50 Monodelphis_domestica VMMMGNVRLAAILLQYGAEPNTPDPTTLTLPVHDAAREGFLDTLMLLHRA 50 Homo_sapiens VMMMGSARVAELLLLHGAEPNCADPATLTRPVHDAAREGFLDTLVVLHRA 50 Pan_troglodytes VMMMGSARVAELLLLHGAEPNCADPATLTRPVHDAAREGFLDTLVVLHRA 50 Bos_taurus -MMMGSARVAELLLLHGADPNCADPATLTRPVHDAAREGFLDTLVALHRA 49 Sus_scrofa VMMMGSTRVAELLLLHGADPNCEDPATLTRPVHDAAREGFLDTLVVLHRA 50 BMD VMMMGSTRVAQLLLLHGADPNCADPVTLTRPVHDAAREGFLDTLVVLHRA 50 CanFam2.0 VMMMGSTRVAQLLLLHGANPNCADPVTLTRPVHDAAREGFLDTLVVLHRA 50 CMT28_FJ542308 VMMMGSTRVAQLLLLHGANPNCADPVTLTRPVHDAAREGFLDTLVVL--- 47 NCF_FJ542309 VMMMGSTRVAQLLLLHGANPNCADPVTLTRPVHDAAREGFLDTLVVL--- 47 Felis_catus VMMMGSARVAELLLLHGADPNCADPATLTRPVHDAAREGFLDTLVVLHRA 50 Equus_caballus VMMMGSVHVAELLLLHGADPNRADPDTLTRPVHDAAREGFL--------- 41 Takifugu_rubripes VMMMGSTRVAQILLDHGADPNVADGTTGATPLHDAARSGFLDTVRLLVRF 50 ****..::* :** :**:.* * * : *:*****.*** Helices helix 7 helix 8 Mus_spretus GARLDVRDAWGRLPLDLAQERGHQDIVRYLR----SAG-CSLCSAGWSLC 95 Mus_musculus GARLDVRDAWGRLPLDLAQERGHQDIVRYLR----SAG-CSLCSAGWSLC 95 Rattus_norvegicus GARLDVRDAWGRLPLDLALERGHHDVVRYLRYLLSSAGNVSRVTDRHNFC 100 Monodelphis_domestica GARLDVRDSWGRLPVDLAEEQGHHLVVAYLR---------EVVRDA---- 87 Homo_sapiens GARLDVRDAWGRLPVDLAEELGHRDVARYLR---AAAGGTRGSNHARIDA 97 Pan_troglodytes GARLDVRDAWGRLPVDLAEELGHRDVARYLR---AAAGGTRGSNHARIDA 97 Bos_taurus GAQLDVRDAWGRLPVDLAEERGHRDVARYLR---AAAEDTEGGSHASADS 96 Sus_scrofa GARLDVRDAWGRLPVDLAEERGHRDVAGYLR---ANAGRTEGGSHARSNS 97 BMD GARLDVRDAWGRLPVDLAEERGHGAVAAYLR---AAAGGTESGSHARTEG 97 CanFam2.0 GARLDVRDAWGRLPVDLAEERGHGAVAAYLR---AAAGGTESGSHARTEG 97 CMT28_FJ542308 -------------------------------------------------- NCF_FJ542309 -------------------------------------------------- Felis_catus GARLDVRDAWGRLPVDLAEERGHRDIVRYLR---ARTGGTGSGSHTGTDG 97 Equus_caballus -------------------------------------------------- Takifugu_rubripes TADPNARDQADRRPVDLARDECHTDVVAFLE---------SL-------- 83 Mus_spretus TAGNVAQ 102 Mus_musculus TAGNVAQ 102 Rattus_norvegicus SS----- 102 Monodelphis_domestica ------- Homo_sapiens AEGPS-- 102 Pan_troglodytes AEGPL-- 102 Bos_taurus AEGPA-- 101 Sus_scrofa GEDPA-- 102 BMD AEGHA-- 102 CanFam2.0 AEGHA-- 102 CMT28_FJ542308 ------- NCF_FJ542309 ------- Felis_catus AEGVA-- 102 Equus_caballus ------- Takifugu_rubripes -------


idues and in the number of GlyAspGlyGly repeats is a nor-mal polymorphism in this species.

p15INK4B, coded by CDKN2B, and the rest of the membersof the inhibitor of kinase 4 (INK4) protein family(p16INK4A, p18INK4C and p19INK4D) have ankyrin repeatsthat play an important role in the folding of the proteinand in intermolecular interactions with other proteins,such as the cyclin-dependent kinases [39]. p15INK4B andp16INK4A are similar proteins that appear to be the result ofa gene duplication event [42]; both have 4 ankyrin repeatsand they appear to function in a similar manner and maybe interchangeable [43]. However, p16INK4A has beenstudied in more detail. It is known, for example, that thisprotein's loops 1 and 2, as well as some of the ankyrinrepeats themselves, interact with CDK6 [38,44-46]. TheGlyAspGlyGly repeats predicted to exist in caninep15INK4B are immediately before helix α1 of the firstankyrin repeat. Hence the polymorphism may not disruptthe folding or function of the protein which is dependenton the ankyrin repeats and the loops connecting them.These GlyAspGlyGly repeats would introduce a region ofsmall glycine residues punctuated by charged aspartatescausing a difference in molecular size at the N-terminus ofthe protein.

p15INK4B is induced by transforming growth factor, beta 1(TGFB1) [47-49]; this induction may be direct, throughthe binding of TGFB1 to a sequence within the promoterof CDKN2B [50]. In a previous study we showed that thebreakpoint of the translocation chromosome t(27;11)involves TGFBR1 [4] which codes for a receptor to TFGB1.However, the tumour still contained a complete codingsequence of the gene. It seems, therefore, that the pathwayis disrupted at the level of p15INK4B and p16INK4A throughthe homozygous deletion of their coding sequences, whilethe possibility still remains that an abnormal p14ARF

could be present.

ConclusionIn fibrosarcomas studied in this work complex rearrange-ments of canine chromosome 11 were observed, causingchanges in genes in the TGFB – p14INK4B pathway. Studyof CDKN2B showed unusual variants present in exon 1 ofthe gene in different breeds of dog, as a result of a simplerepeat sequence. No instances of missense or frameshift-ing mutations were observed. However, it is possible thatthe genomic plasticity of CDKN2B observed here is con-nected with the rather high frequency of cancers in thedomestic dog.

MethodsTumours and blood samplesThe two tumours, corresponding to two dogs name-codedLE and ME, have been described previously [4]. Both are

fibrosarcomas from adult Labrador retriever females fromthe pet population. Blood samples were obtained fromexcess material used for the purpose of clinical diagnosticstudies of dogs admitted to the Queens Veterinary SchoolHospital of the University of Cambridge.

Fluorescence-in-situ HybridisationChromosome paints for seven colour FISH and othercombinations of single colour canine chromosome paintswere prepared and used as described previously [51].BACs were from the RPCI-81 canine BAC library [52], andDNA's from them were labeled with biotin-dUTP or dig-oxigenin-dUTP by nick translation. For hybridization,300 ng of labeled DNA in 1 μl TE representing one chro-mosome paint probe mix, or 50 ng of labeled BAC DNA,was mixed with 14 μl hybridization buffer (55% forma-mide,11% dextran sulphate, 2.2 × SSC, 45 mM sodiumphosphate pH7,1.1× Denhardt's solution (Sigma), 0.5mM EDTA, 45 μg/ml canine Cot-1 DNA), heated at 70Cfor 10 minutes, then left 20–30 minutes at 37°C to allowpre-annealing of the Cot-1 DNA. Metaphase preparationson glass slides were denatured, dried and hybridized tothe denatured probes. Hybridization was overnight at37°C in a humid chamber. Slides were washed two × 5mins in 50% formamide/50% 2 × SSC at 45C followed bytwo × 5 mins in 2 × SSC at 45°C and a final wash in 4 ×SSC, 0.01% Tween20 before application of fluorochrome-conjugated antibodies.

Biotin labeled probes were visualized using Cy3-avidin(single colour FISH) or Cy5-avidin (multi-colour FISH)(1:500 dilution, Amersham). FITC labeled probes werevisualized using sequential application and washing ofrabbit anti-FITC (1:200) and FITC-labeled goat anti-rabbitantibodies (1:100). After 20 minutes incubation at 37°C,excess antibody was washed off by three 5 minute washesin 4 × T at 42°C. Slides were mounted with Vectashieldcontaining DAPI and images were captured as before [53].Fluorescence signals were captured separately as 8-bitblack and white images through appropriate excitation fil-ters, normalized, and merged into a 24-bit colour image.10 – 15 metaphases were captured for each probe mix.

DNA extraction form bloodFor one of the patients (LE) blood was available and DNAwas extracted using the QIAamp DNA Blood Mini kit(QIAGEN) according to the manufacturer's instructions.This same extraction procedure was used for the bloodsused to study the CDKN2B exon 1 polymorphism in 18breeds.

Loss of heterozygosity analysisFor individual LE, for which tumour and blood DNA wasavailable, loss of heterozygosity analysis was performed.Microsatellite markers were chosen along the canine chro-



mosome 11; the location of non CAMC markers and theirprimer sequences appear in the dog reference genome inEnsembl [7]. To fill the gaps between adjacent markers,and to increase the density in some regions of interest,such as those around the CDKN2B-CDKN2A locus andthe TFGBR1 gene, additional microsatellites were identi-fied from the dog reference sequence: appropriate contigsequences were analysed with Tandem Repeat Finder ver-sion 4.00 [54] and primers were designed manually orusing Primer3 [55,56]. These markers carry the prefixCAMC in the name. All PCR reactions had a final volumeof 10 μl, with 1 to 10 ng of genomic DNA as template, 5U of Taq polymerase (Invitrogen), a final concentration of0.2 pmol/μl for each primer, 1.5 mM of MgCl2 and 0.25mM of each dNTP. For markers with the CAMC prefix, thePCR products were labelled indirectly using a two-stepamplification procedure. For these markers, the forwardmarker-specific primer had a T7 universal sequence (5'-TAATACGACTCACTATAGGG-3') added to its 5' end.These markers, with forward and reverse primer sequences(omitting the T7 sequence from the forward primer), are:CAMC11.001 (AAATGGTCCATCAGAG, ACTAGCTAA-GAACTTCATGG); CAMC11.002 (AGTGCTTTGAT-GCTGA, GAAAGACCTACATTAGTACC); CAMC11.003(ATAAGACCAAACCTAC, TCTCATGTTTCTGAAGAGTG);CAMC11.004 (AAGTAGCCATACTAAT, CTCAGACCT-TATCTATTTGG); CAMC11.005 (ATGGGTATTAAAGACG,CCAAACTGAACCAACATGAG); CAMC11.006 (AACACT-CAGTCAAGCC, ATCGGACTCCTTCCATGCTG);CAMC11.007 (CTTGGATAAGCTCTGC, TTCTGGACAG-TACTACATTC); CAMC11.008 (GGAGGGGGATT-GGTTTTGAT, ATGCTCACTCACTCGCATGT);CAMC11.009 (CCTGTTGGAACTGGAGCTTT, TTTGGGA-TAATCTAAAGCAAATC); CAMC11.010 (GGGTACCT-TCTTTCGGCATT, CATAGCTGACTCCCTTGAAG);CAMC11.011 (CCCTCATACCACGGCTTTTA, ATGAC-CAAAACTGCCCAGAG); CAMC11.012 (TTCTTCTTT-GGGAGTTGCACA, TTCAGCTCCCTTGGAAACTC);CAMC11.013 (ATGGTAGATGGAGGACCTGA, CAGGAT-GTGTGAAGGGGAG); CAMC11.014 (GGCAGAGGGT-GGGTAAAATA, GCCGCCCTGTATGCCTTTT);CAMC11.015 (ACATGTGAAGGCAACCATTTG, TTGCT-TAGACAGTATGAAATATG); CAMC11.016 (TTATCCCT-GGAAACCCAACAT, TGGCCAAGGTTACACAGTAG);CAMC11.017 (GATGGGACAAGTGTCAAAGG, TTGTC-CTCTGAGCAAGTCTG); CAMC11.018 (CAG-GAGCCCACTCCTTTTTA, GAGTGACTGTGCGTGTGTG);CAMC11.019 (GCACATGAGGACCCTTCAAT, ATGTCAT-GGAAAACTGCAAGC); CAMC11.020 (AGTAAACACTAC-CACATCTGG, TCAGGTGGAGCCAGGTTTTA);CAMC11.021 (ATCATGGAACCCAGAGCAAC,AAGCCAGCTCATCAAGGAGA); CAMC11.022 (TCCT-GAGAAAGGCCTGGATA, GCCCGTCAAGTTAGTGAGG);CAMC11.023 (CTGCCAACTCCTCCTCTGT, CCCTC-CCAACTGTTCCAAAT); CAMC11.024 (AACAGCAG-

GACAAAGTCTGC, ATATCATTCGTGGCCCTTCC);CAMC11.026 (TCATGGGTCATGAGATGGAC, GCCCT-CATGAATGGGATTAG); CAMC11.027 (CACA-GAATAACTCAATAGGTTG,ACTTCTGTGAAGTGCCTTATG); CAMC11.029 (GGT-TCAAGTTCAGAATGCTTG, GTTTAGCGT-TAGCGCCTGC). For the first PCR these unlabelledprimers were used; 1 μl of these products were then usedas template for a second PCR reaction with a T7 primerhaving a D2, D3 or D4 label (Proligo) on its end. The firstPCR reaction was a touch-down PCR with an initialannealing temperature of 66°C; this temperature wasdecreased by 1°C for 12 cycles, followed by 30 cycles withan annealing temperature of 54°C. For the second PCR 25cycles were performed with an annealing temperature of54°C. For all PCRs, each step had a duration of 20 sec. Formarkers whose name does not have the prefix CAMC, thePCR amplifications were performed with a single reactionusing forward primers labelled with D2, D3 or D4 (Pro-ligo). After PCR amplification, regardless of whether oneor two step reactions had been performed, all PCR prod-ucts were diluted 1:8 to 1:12 using sterile water. Twomicrolitres of each diluted product were mixed with 0.15μl of D1-labelled GenomeLab DNA size standard 600(Beckman Coulter) and 30 μl of GenomeLab SampleLoading Solution containing formamide (Beckman Coul-ter). The products were then run in a CEQ8000 sequencer(Beckman Coulter) and genotypes were retrieved with theinstrument's Fragment Analysis software. This same pro-gram was used to determine loss of heterozygosity, bycomparing the peaks obtained from the blood sample andfor the matching tumour. Loss of heterozygosity wasnoted when either peak showed a greater than 30% loss ofsignal.

CDKN2B and CDKN2A sequencingPrimers were designed for studying CDKN2B andCDKN2A exonic sequences plus the flanking intronicbases. The primer names, sequences (5' to 3'), starting andending annealing temperatures for touchdown PCR, andPCR product sizes were as follows. For CDKN2B exon 1primers 1729 (GAAACAGGGAGGCGAGTGAC) and 1730(CGCAGATACGTCGGCGAAGC), 70°C to 58°C, 388 bp;for CDKN2B exon 2 primers 1731 (GAAATGGTCCACCT-GTCCCTG) and 1732 (CACCGTGACTCAAGTCTCCTG),72°C to 60°C, 470 bp; for CDKN2A exon 1b primers2257 (GAGCTTCCACCCCTAGAAAC) and 2258(CGGCTCCGAGATCGGAGG), 72°C to 60°C, 526 bp;and for CDKN2A exon 2 primers 1727 (CTTGTAGCG-GCATCTGCATGG) and 1728 (TGCTCTGGGCTGCG-GAAG), 72°C to 62°C, 459 bp. CDKN2B and CDKN2Asequences are GC-rich, so they were PCR-amplified withAccuPrime GC-Rich DNA polymerase (Invitrogen) usingBuffer A for that enzyme. PCRs were done in 40 μl, using6 to 30 ng of genomic DNA, a final concentration of



02.pmol/μl for each primer and 1.6 U of the polymerase.All amplifications were done using touchdown-PCR. PCRproducts, except for CDKN2B exon 1, were cleaned withthe QIAquick PCR purification kit (QIAGEN), sequencedwith the GenomeLab DTCS kit (Beckman Coulter) andran in a CEQ8000 sequencer. Sequences were analysedand aligned against the canine genomic referencesequences using the Sequence Analysis and SequenceInvestigator modules on the sequencer. Differences withrespect to the reference sequence were confirmed bysequencing the complementary strand. CDKN2B exon 1PCR fragments from tumour samples were cloned andthen sequenced. AccuPrime's GC-rich polymerase proof-reading activity yields blunt-ended products; to clone thefragments into the PCR4-TOPO vector of the TOPO TACloning Kit for Sequencing (Invitrogen) a 3' A-overhangwas incorporated to the PCR products by incubating themwith 1 μl of Taq polymerase (Invitrogen) at 72°C for 10min. Sequencing reactions with the GenomeLab DTCS kitwere performed according to the manufacturer's protocolfor plasmid templates except that betaine (Sigma) wasadded to a final concentration of 1 M. The referencesequences used were CanFam 2.0, assembly May 2006,Genebuild Sep 2008 [7,8] for the canine genome; [EMBL:FM946072] for CDKN2B and [EMBL: FM883643] forp14ARF/CDKN2A. Polymorphisms and mutations in thesesequences are described following the recommendednomenclature [57,58].

CDKN2B exon 1 polymorphism searchTo determine the CDKN2B exon 1 alleles present in a col-lection of dog DNAs of different breeds, the exon wasamplified, along with the flanking intronic sequences,using a D4 (Sigma) labelled forward primer 1729 and anunlabelled reverse primer 1730. The length of the prod-uct, according to the reference sequence in CanFam2.0 is388 bp [7]. PCRs were performed using the KAPA2GRobust PCR Kit (Kapabiosystems, Cape Town, SouthAfrica), with buffer B and Kapa Enhancer 1, in a volumeof 10 μl, with a minimum of 8 ng of template and a touch-down-PCR starting with an annealing temperature of72°C, and decreasing by 1°C per cycle to 59°C, followedby 25 cycles with an annealing temperature of 58°C.Annealing, extension (at 72°C) and denaturation (at95°C) were 20 sec long each. The products were diluted1:8 with sterile water and loaded on a CEQ8000sequencer (Beckman Coulter) to determine the size of thePCR products with the Fragment Analysis module of theinstrument; 18.25% of the samples were retested to con-firm the results. 22.63% of the samples, representing thedifferent allele sizes observed, were amplified again fromgenomic DNA and sequenced directly with the Genom-eLab DTCS kit following the procedure recommended bythe manufacturer for GC-rich sequences. Cleaned prod-ucts were separated on the CEQ8000 at 60°C and results

analysed as in the sequences described in the previous sec-tion. Multiple alignments of nucleotide and predictedamino acid sequences were done with ClustalW2 [59] atthe EMBL-EBI website [60] or using JalView 2.3 [61] andthen manually edited. GenBank accession numbers forp15INK4B amino acid sequences used for the alignmentswere [Xenopus tropicalis GenBank: AAH75575.1, Rattusnorvegicus GenBank: EDL97746.1, Mus musculus GenBank:AAH02010.1, Bos taurus GenBank: NP_001069362,Macaca mulatta GenBank: XP_001107263.1, Pan troglo-dytes GenBank: ENSPTRP00000035618, Homo sapiensGenBank: P42772]. The sequences for p16INK4A were [Tak-ifugu rubripes GenBank: CAC12808.1, Monodelphis domes-tica GenBank: AAC23669.1, Bos taurus GenBank:XP_612365.1, Equus caballus GenBank: AAC97110.1, Susscrofa GenBank: CAB65454.1, Mus spretus GenBank:AAD00236.1, Rattus norvegicus GenBank: AAL76339.1,Mus musculus UniProt: P51480-1, Felis catus GenBank:BAA33540.1, Pan troglodytes GenBank: XP_520513.1,Homo sapiens GenBank: NP_478104.1, CMT28 GenBank:FJ542308.1, NCF GenBank: FJ542309.1] and for p14ARF

[Mesocricetus auratus GenBank: AAN75824.1, Rattus nor-vegicus GenBank: AAL76336.1, Mus sp. GenBank:AAB35770.1, Homo sapiens GenBank: NP_478102.1].

In the flat-coated retrievers allele distribution betweencases and controls was compared using a Monte Carlosimulation of the chi-square test and grouping togethercolumns with expected values lower than 5, as imple-mented in the T2 test of the program Clump [62]; the levelof significance was determined by performing 10000 sim-ulations.

Competing interestsThe authors declare that they have no competing interests.

Authors' contributionsJAH performed the LOH study, the sequencing of tumoursamples, the DNA extraction and sequencing of bloodsamples, the alignments and bioinformatics analyses andwrote the manuscript. BM established the primary cell cul-tures, carried out the cytogenetic studies and made thechromosome paints. CQ performed the BAC mapping.PCMO flow-sorted the chromosomes. TH collected thebiological samples and maintained the information data-base associated with it. SH performed the pathologicaldiagnosis of the tumours and other biopsy samples. MAFSparticipated in the design of the study and in the interpre-tation of the results. JMD participated in the design of theproject and in the collection of samples. DRS conceivedand directed the project, interpreted the cytogenetic andBAC mapping results, analysed the sequencing data andparticipated in writing the manuscript. All authors readand approved the final manuscript.


http://www.ebi.ac.uk/cgi-bin/dbfetch?FM946072

http://www.ebi.ac.uk/cgi-bin/dbfetch?FM883643

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=AAH75575.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=EDL97746.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=AAH02010.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=NP_001069362

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=XP_001107263.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=ENSPTRP00000035618

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=P42772

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=CAC12808.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=AAC23669.1


http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=AAC97110.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=CAB65454.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=AAD00236.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=AAL76339.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=BAA33540.1


http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=NP_478104.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=FJ542308.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=FJ542309.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=AAN75824.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=AAL76336.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=AAB35770.1

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=NP_478102.1


Additional material

AcknowledgementsThese studies were supported by the Pet Plan ChariTable Trust and Cancer Research UK grant reference C8876/A4012. BM received a Domestic Research Studentship from Cambridge University. Some of the DNA sam-ples used in this study were kindly provided by Keiko Miyadera, Veterinary Medicine, University of Cambridge. This project benefited from the use of the University of Cambridge's federated computational grid (CamGrid).

References1. Withrow SJ, Powers BE, Straw RC, Wilkins RM: Comparative

aspects of osteosarcoma. Dog versus man. Clin Orthop Relat Res1991, 270:159-168.

2. Vail DM, MacEwen EG: Spontaneously occurring tumors ofcompanion animals as models for human cancer. Cancer Invest2000, 18:781-792.

3. Hendrick MJ, Mahaffey EA, Moore FM, Vos JH, Walder EJ: Histologicalclassification of mesenchymal tumors of skin and soft tissues of domesticanimals Washington, D.C: American Registry of Pathology; 1998.

4. Sargan DR, Milne BS, Aguirre Hernandez J, O'Brien PC, Ferguson-Smith MA, Hoather T, Dobson JM: Chromosome rearrange-ments in canine fibrosarcomas. J Hered 2005, 96:766-773.

5. Guyon R, Lorentzen TD, Hitte C, Kim L, Cadieu E, Parker HG, Qui-gnon P, Lowe JK, Renier C, Gelfenbeyn B, Vignaux F, DeFrance HB,Gloux S, Mahairas GG, Andre C, Galibert F, Ostrander EA: A 1-Mbresolution radiation hybrid map of the canine genome. ProcNatl Acad Sci USA 2003, 100:5296-5301.

6. Gil J, Peters G: Regulation of the INK4b-ARF-INK4a tumoursuppressor locus: all for one or one for all. Nat Rev Mol Cell Biol2006, 7:667-677.

7. Ensembl [http://www.ensembl.org]8. Lindblad-Toh K, Wade CM, Mikkelsen TS, Karlsson EK, Jaffe DB,

Kamal M, Clamp M, Chang JL, Kulbokas EJ 3rd, Zody MC, et al.:Genome sequence, comparative analysis and haplotypestructure of the domestic dog. Nature 2005, 438:803-819.

9. Bignell GR, Santarius T, Pole JC, Butler AP, Perry J, Pleasance E,Greenman C, Menzies A, Taylor S, Edkins S, Campbell P, Quail M,Plumb B, Matthews L, McLay K, Edwards PA, Rogers J, Wooster R,Futreal PA, Stratton MR: Architectures of somatic genomicrearrangement in human cancer amplicons at sequence-level resolution. Genome Res 2007, 17:1296-1303.

10. Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson DA: Dele-tions of the cyclin-dependent kinase-4 inhibitor gene in mul-tiple human cancers. Nature 1994, 368:753-756.

11. Sharpless NE: INK4a/ARF: a multifunctional tumor suppressorlocus. Mutat Res 2005, 576:22-38.

12. Ruas M, Peters G: The p16INK4a/CDKN2A tumor suppressorand its relatives. Biochim Biophys Acta 1998, 1378:F115-177.

13. Reef S, Zalckvar E, Shifman O, Bialik S, Sabanay H, Oren M, Kimchi A:A short mitochondrial form of p19ARF induces autophagyand caspase-independent cell death. Mol Cell 2006, 22:463-475.

14. Reef S, Shifman O, Oren M, Kimchi A: The autophagic inducersmARF interacts with and is stabilized by the mitochondrialp32 protein. Oncogene 2007, 26:6677-6683.

15. Ueda Y, Koya T, Yoneda-Kato N, Kato JY: Small mitochondrialARF (smARF) is located in both the nucleus and cytoplasm,induces cell death, and activates p53 in mouse fibroblasts.FEBS Lett 2008, 582:1459-1464.

16. Robertson KD, Jones PA: Tissue-specific alternative splicing inthe human INK4a/ARF cell cycle regulatory locus. Oncogene1999, 18:3810-3820.

17. Lin YC, Diccianni MB, Kim Y, Lin HH, Lee CH, Lin RJ, Joo SH, Li J,Chuang TJ, Yang AS, Kuo HH, Tsai MD, Yu AL: Humanp16gamma, a novel transcriptional variant of p16(INK4A),coexpresses with p16(INK4A) in cancer cells and inhibitscell-cycle progression. Oncogene 2007, 26:7017-7027.

18. Krimpenfort P, Quon KC, Mooi WJ, Loonstra A, Berns A: Loss ofp16Ink4a confers susceptibility to metastatic melanoma inmice. Nature 2001, 413:83-86.

19. Sharpless NE, Bardeesy N, Lee KH, Carrasco D, Castrillon DH,Aguirre AJ, Wu EA, Horner JW, DePinho RA: Loss of p16Ink4awith retention of p19Arf predisposes mice to tumorigenesis.Nature 2001, 413:86-91.

20. Kamijo T, Zindy F, Roussel MF, Quelle DE, Downing JR, Ashmun RA,Grosveld G, Sherr CJ: Tumor suppression at the mouse INK4alocus mediated by the alternative reading frame productp19ARF. Cell 1997, 91:649-659.

21. Sharpless NE, Ramsey MR, Balasubramanian P, Castrillon DH,DePinho RA: The differential impact of p16(INK4a) orp19(ARF) deficiency on cell growth and tumorigenesis. Onco-gene 2004, 23:379-385.

22. Liu Q, Neuhausen S, McClure M, Frye C, Weaver-Feldhaus J, GruisNA, Eddington K, Allalunis-Turner MJ, Skolnick MH, Fujimura FK, etal.: CDKN2 (MTS1) tumor suppressor gene mutations inhuman tumor cell lines. Oncogene 1995, 10:1061-1067.

23. Lohmann DR: RB1 gene mutations in retinoblastoma. HumMutat 1999, 14:283-288.

24. Drexler HG: Review of alterations of the cyclin-dependentkinase inhibitor INK4 family genes p15, p16, p18 and p19 inhuman leukemia-lymphoma cells. Leukemia 1998, 12:845-859.

25. Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger PC, BaylinSB, Sidransky D: 5' CpG island methylation is associated withtranscriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat Med 1995, 1:686-692.

26. Rocco JW, Sidransky D: p16(MTS-1/CDKN2/INK4a) in cancerprogression. Exp Cell Res 2001, 264:42-55.

27. Orlow I, Drobnjak M, Zhang ZF, Lewis J, Woodruff JM, Brennan MF,Cordon-Cardo C: Alterations of INK4A and INK4B genes inadult soft tissue sarcomas: effect on survival. J Natl Cancer Inst1999, 91:73-79.

28. Koenig A, Bianco SR, Fosmire S, Wojcieszyn J, Modiano JF: Expres-sion and significance of p53, rb, p21/waf-1, p16/ink-4a, andPTEN tumor suppressors in canine melanoma. Vet Pathol2002, 39:458-472.

29. Levine RA, Fleischli MA: Inactivation of p53 and retinoblastomafamily pathways in canine osteosarcoma cell lines. Vet Pathol2000, 37:54-61.

30. Fosmire SP, Thomas R, Jubala CM, Wojcieszyn JW, Valli VE, GetzyDM, Smith TL, Gardner LA, Ritt MG, Bell JS, Freeman KP, GreenfieldBE, Lana SE, Kisseberth WC, Helfand SC, Cutter GR, Breen M, Modi-ano JF: Inactivation of the p16 cyclin-dependent kinase inhib-itor in high-grade canine non-Hodgkin's T-cell lymphoma.Vet Pathol 2007, 44:467-478.

31. DeInnocentes P, Agarwal P, Bird RC: Phenotype-rescue of cyclin-dependent kinase inhibitor p16/INK4A defects in a spontane-ous canine cell model of breast cancer. J Cell Biochem 2009,106:491-505.

32. Korgaonkar C, Zhao L, Modestou M, Quelle DE: ARF functiondoes not require p53 stabilization or Mdm2 relocalization.Mol Cell Biol 2002, 22:196-206.

33. Llanos S, Clark PA, Rowe J, Peters G: Stabilization of p53 byp14ARF without relocation of MDM2 to the nucleolus. NatCell Biol 2001, 3:445-452.

34. Kim SH, Mitchell M, Fujii H, Llanos S, Peters G: Absence ofp16INK4a and truncation of ARF tumor suppressors inchickens. Proc Natl Acad Sci USA 2003, 100:211-216.

Additional file 1Distribution of CDKN2B exon 1 alleles in various dog breeds. Cells show number of individuals for the corresponding genotypes and alleles. In the heading, genotypes and alleles are described in a simplified way (e.g., allele g. [109GGC[2];124GGGGACGGCGGC[3] appears as [2][3], while the heterozygous combination of g. [109GGC[2];124GGGGACGGCGGC[3] and g. [109GGC[5];124GGGGACGGCGGC[3] is shown as [2][3][5][3]. In the flat-coated retrievers, HS denotes histiocytic sarcoma).Click here for file[http://www.biomedcentral.com/content/supplementary/1746-6148-5-27-S1.xls]


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35. Chen YJ, Chang JG, Shih LS, Chen PH, Endo M, Whang-Peng J, ChenYM: Frequent detection of aberrant RNA transcripts of theCDKN2 gene in human gastric adenocarcinoma. Int J Cancer1997, 71:350-354.

36. Cho JW, Jeong YW, Han SW, Park JB, Jang BC, Baek WK, Kwon TK,Park JW, Kim SP, Suh MH, Suh SI: Aberrant p16INK4A RNA tran-scripts expressed in hepatocellular carcinoma cell lines reg-ulate pRb phosphorylation by binding with CDK4, resultingin delayed cell cycle progression. Liver Int 2003, 23:194-200.

37. Baumgartner R, Fernandez-Catalan C, Winoto A, Huber R, Engh RA,Holak TA: Structure of human cyclin-dependent kinase inhib-itor p19INK4d: comparison to known ankyrin-repeat-con-taining structures and implications for the dysfunction oftumor suppressor p16INK4a. Structure 1998, 6:1279-1290.

38. Li J, Byeon IJ, Ericson K, Poi MJ, O'Maille P, Selby T, Tsai MD: Tumorsuppressor INK4: determination of the solution structure ofp18INK4C and demonstration of the functional significanceof loops in p18INK4C and p16INK4A. Biochemistry 1999,38:2930-2940.

39. Li J, Mahajan A, Tsai MD: Ankyrin repeat: a unique motif medi-ating protein-protein interactions. Biochemistry 2006,45:15168-15178.

40. Lohi H, Young EJ, Fitzmaurice SN, Rusbridge C, Chan EM, VervoortM, Turnbull J, Zhao XC, Ianzano L, Paterson AD, Sutter NB,Ostrander EA, Andre C, Shelton GD, Ackerley CA, Scherer SW,Minassian BA: Expanded repeat in canine epilepsy. Science 2005,307:81.

41. Fondon JW 3rd, Garner HR: Molecular origins of rapid and con-tinuous morphological evolution. Proc Natl Acad Sci USA 2004,101:18058-18063.

42. Gilley J, Fried M: One INK4 gene and no ARF at the Fugu equiv-alent of the human INK4A/ARF/INK4B tumour suppressorlocus. Oncogene 2001, 20:7447-7452.

43. Krimpenfort P, Ijpenberg A, Song JY, Valk M van der, Nawijn M,Zevenhoven J, Berns A: p15Ink4b is a critical tumour suppres-sor in the absence of p16Ink4a. Nature 2007, 448:943-946.

44. Byeon IJ, Li J, Ericson K, Selby TL, Tevelev A, Kim HJ, O'Maille P, TsaiMD: Tumor suppressor p16INK4A: determination of solu-tion structure and analyses of its interaction with cyclin-dependent kinase 4. Mol Cell 1998, 1:421-431.

45. Russo AA, Tong L, Lee JO, Jeffrey PD, Pavletich NP: Structuralbasis for inhibition of the cyclin-dependent kinase Cdk6 bythe tumour suppressor p16INK4a. Nature 1998, 395:237-243.

46. Li J, Poi MJ, Qin D, Selby TL, Byeon IJ, Tsai MD: Tumor suppressorINK4: quantitative structure-function analyses of p18INK4Cas an inhibitor of cyclin-dependent kinase 4. Biochemistry 2000,39:649-657.

47. Hannon GJ, Beach D: p15INK4B is a potential effector of TGF-beta-induced cell cycle arrest. Nature 1994, 371:257-261.

48. Sandhu C, Garbe J, Bhattacharya N, Daksis J, Pan CH, Yaswen P, KohJ, Slingerland JM, Stampfer MR: Transforming growth factor betastabilizes p15INK4B protein, increases p15INK4B-cdk4 com-plexes, and inhibits cyclin D1-cdk4 association in humanmammary epithelial cells. Mol Cell Biol 1997, 17:2458-2467.

49. Reynisdottir I, Polyak K, Iavarone A, Massague J: Kip/Cip and Ink4Cdk inhibitors cooperate to induce cell cycle arrest inresponse to TGF-beta. Genes Dev 1995, 9:1831-1845.

50. Li JM, Nichols MA, Chandrasekharan S, Xiong Y, Wang XF: Trans-forming growth factor beta activates the promoter of cyclin-dependent kinase inhibitor p15INK4B through an Sp1 con-sensus site. J Biol Chem 1995, 270:26750-26753.

51. Milne BS, Hoather T, O'Brien PC, Yang F, Ferguson-Smith MA, Dob-son J, Sargan D: Karyotype of canine soft tissue sarcomas: amulti-colour, multi-species approach to canine chromosomepainting. Chromosome Res 2004, 12:825-835.

52. Li R, Mignot E, Faraco J, Kadotani H, Cantanese J, Zhao B, Lin X, Hin-ton L, Ostrander EA, Patterson DF, de Jong PJ: Construction andcharacterization of an eightfold redundant dog genomic bac-terial artificial chromosome library. Genomics 1999, 58:9-17.

53. Yang F, O'Brien PC, Milne BS, Graphodatsky AS, Solanky N, TrifonovV, Rens W, Sargan D, Ferguson-Smith MA: A complete compara-tive chromosome map for the dog, red fox, and human andits integration with canine genetic maps. Genomics 1999,62:189-202.

54. Benson G: Tandem repeats finder: a program to analyze DNAsequences. Nucleic Acids Res 1999, 27:573-580.

55. Rozen S, Skaletsky J: Primer3 on the WWW for general usersand for biologist programmers. In Bioinformatics Methods and Pro-tocols: Methods in Molecular Biology Edited by: Krawetz S, Misener S.Totowa, NJ: Humana Press; 2000:365-386.

56. Primer3 (v. 0.4.0) [http://frodo.wi.mit.edu/primer3/]57. HGVS, nomenclature for the description of sequence varia-

tions [http://www.hgvs.org/mutnomen/]58. den Dunnen JT, Antonarakis SE: Mutation nomenclature exten-

sions and suggestions to describe complex mutations: a dis-cussion. Hum Mutat 2000, 15:7-12.

59. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA,McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, ThompsonJD, Gibson TJ, Higgins DG: Clustal W and Clustal X version 2.0.Bioinformatics 2007, 23:2947-2948.

60. EMBL-EBI, ClustalW2 [http://www.ebi.ac.uk/Tools/clustalw2/]61. Clamp M, Cuff J, Searle SM, Barton GJ: The Jalview Java alignment

editor. Bioinformatics 2004, 20:426-427.62. Sham PC, Curtis D: Monte Carlo tests for associations between

disease and alleles at highly polymorphic loci. Ann Hum Genet1995, 59(Pt 1):97-105.






















































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Disruption of chromosome 11 in canine fibrosarcomas highlights an unusual variability of CDKN2B in dogs